Mastitis is a general term that describes inflammation in the milk glands and tissue lining the mammary gland/udder. Inflammation is a cellular- and organ-specific response to injury that can arise from a number of environmental events and pathogenic agents. Mastitis can be induced by physical injury (such as that caused by the milking apparatus), and from bacterial and fungal infections. Infections of the nipples, ducts and udders might be caused by mycoplasma, pseudomonas, staphylococcus and streptococcus, as well as coliforms such Escherichia coli. Probably more than a dozen different bacterial species, in addition to yeast and fungi, can cause mastitis in cow, sheep and goat. Yeast and fungal infections yield a sub-clinical form of infection and therefore may go undetected. However, it is not known if yeast/fungi can aggravate infection caused by coincident bacterial infection.
The living environment of dairy cows is conducive to infection, since the soil and manure are rich sources of bacteria. The animals are in frequent contact with the soil, and this is evident when one compares natural fluctuations in the sub-clinical infections during wet and dry seasons; wet environments cause higher levels of somatic cell count (SCC) values. Furthermore, the milking process is itself highly conducive to cross contamination. Therefore, animal management procedures play a key role in controlling the incidence of mastitis. Separation of infected animals, clean handling techniques, and well fitting vacuum suction cups can play an important role in minimizing the incidence of infection. Treatment of the affected animal includes antibiotics to eliminate the bacteria, and anti-inflammatory agents to reduce swelling.
In cows, the four teats drain separate compartments (quarters) in the udder, and the infection can migrate from one quarter to another in a single animal. Additionally, animal-to-animal and human contact can also extend the infection to other animals in the milk line. These issues are of considerable concern, since the milking process typically results in the collection of milk from many animals (50-200) in a common reservoir (bulk tank). Not surprisingly, a single heavily infected animal can compromise the quality of the entire milk output from a dairy facility. Culling chronically infected, unresponsive animals is a prudent measure. However, reduced milk output, poor milk quality, veterinary and health issues have a significant negative impact on the viability and productivity in dairy farms. In this regard, the technologies developed and described herein are expected to contribute to improving early diagnostics, minimizing cross-contamination, sequestering problematic animals, and monitoring efficacy of treatment.
Mastitis is characterized as clinical or pre-clinical mastitis. Sub-clinical mastitis is not visually detected (e.g., redness, soreness, swelling), and milk quality is not severely diminished. However, some changes may be observed, including clots and runny consistency of the milk. Redness, soreness and swelling of the teats are occasionally observed. Clinical mastitis can be mild or severe, based on the level of SCC in the milk. A heavily infected animal will display significant redness, soreness, and a hard teat, which will necessitate antibiotic treatment. The onset of mastitis can be rapid, and cause impaired breathing, failure to eat, and significantly increased body temperature. A systemic infection is considered grave, and the animal is removed from the milk line, and treated with antibiotics for approximately 3 days. Following the treatment, the animal is kept off the milk line until antibiotic levels in the milk are reduced. Some animals may tolerate chronic sub-clinical mastitis, although the extent of this occurrence and its effect of milk output and quality have not been carefully determined, because there is no straightforward way to measure it. Therefore, there is a need for detecting the early stage of infection to permit prompt removal of affected individuals before the bulk reservoir is affected. Because chronically affected individually oscillate between high baseline of sub-clinical infection and full-blown infection, these candidates require constant monitoring, which is not possible with conventional methods, but can be easily achieved with the Invention described here.
The standard assay for measuring mastitis in the dairy industry is to count the number of cells (originating from the cow), in the milk. This estimate is termed the somatic cell count (SCC). Specifically, the SCC reflects the levels of cells, including immune cells, such as leukocytes that are released from the lining and tissues of the udder of the infected animal, into the udder cavity. Although these cells are present in the milk, it is difficult to estimate their levels due to the difficulty of visualizing clear cells in the turbid milk suspension. The number of somatic cells in a given volume of milk (typically 1 ml) provides a semi-quantitative estimate of the degree of infection, because unaffected animals typically have low SCC levels. The SCC level may also be confounded by high levels of a pathogen, including bacteria, yeast and fungi, each of which is a causative agent in mastitis. There are a number of limitations of the SCC type assay. Due to turbidity, milk cell counts cannot be made, and instead the samples are tested at remote laboratories using independent measurements. The time lapse between the collection of a milk sample and the transmittal of the cell count information to the dairy farmer can exceed one month. This extraordinary delay in providing key information to the field agent or dairy farmer severely limits prompt action that might otherwise curtail the transmission of infection to unaffected animals. High level of SCC in the bulk tank deteriorates milk quality, and garners a lower price for the farmer. Furthermore, mastitis reduces milk output in the affected animal, and increases the likelihood of more frequent infections. Surprisingly, infected animals are only treated for a fixed term with antibiotics, and SCC levels are not typically monitored to confirm successful outcome. Consequently, an animal might be treated excessively, or not sufficiently. In the latter case, animals that respond poorly, or slowly to the antibiotic regimen, are likely to become chronic offenders that become drug resistant, yield poor quality of milk, incur large veterinarian expenses, eventually leading to the culling of the animal.
Federal and State guidelines allow up to about 7.5×105 somatic cells/ml of raw milk. This is the upper limit, and milk quality is negatively affected at such high SCC levels. Typical values in the bulk tank may range from 2-4×105 SCC. The level permitted by the European dairy industry is more stringent (4×105 somatic cells/ml).
There are currently two broadly available mastitis tests for monitoring milk quality. One assay, called the Somatic Cell Count (SCC) determines the level of somatic cells in the milk. The weaknesses of the SCC assay include inaccuracy, since it is negatively influenced by the presence of pathogens (the primary cause of mastitis) in the milk; insensitivity as it only provides a threshold value of the levels of somatic cells; and failure to provide early-diagnostic information, because the results are provided to the farm up to a month after the initial acquisition of milk samples. Moreover, an individual infected animal is not identified because SCC levels are typically measured in the bulk milk reservoir, which can contain milk from 50-100 cows. Therefore, this compromises the quality of milk in the bulk reservoir and delays detection of the affected animal. Moreover, advanced mastitis necessitates more aggressive treatment, prolonged withdrawal of the animal from the milk line, and a higher probability of generating a chronically affected individual, all of which represent significant economic liabilities to the farm. The SCC assay is hindered by the expense and delay of testing samples at a remote laboratory. Significantly, the delay prevents the implementation of prompt remedial action. Generally, the farmer/field agent recognizes symptoms in an affected animal and removes it from the milk line. However, this represents an action after the infection has occurred.
The second assay is termed the California Mastitis Test (CMT). In this method, milk from each quadrant of the udder is deposited into each of four shallow receptacles, to which a proprietary solution is added. Gentle mixing results in clumping of mastitis-positive samples. This is an imprecise assay that does not give any quantitative measurement of the level of infection. Moreover, the assay is quite insensitive, and does not detect lower levels of persistent mastitis.
While other assays have been suggested, these assays have not been developed for commercial use. For example, Pyörälä ((2003) Vet. Res. 34:565-578) suggests that ATP has a strong positive correlation with SCC and has been considered as an alternative to SCC as an indicator of mastitis. Similarly, GB 2001434 indicates that ATP levels correlate with somatic cell count numbers and can be used to determine the hygiene of milk or the health condition of cows. However, neither of these assays detects the level of proteasome activity via a proteasomal substrate or hydrolysis of ATP.
Clinical mastitis causes greater than $2 billion in directly attributable losses for the dairy industry. However, this is an underestimate, because the financial loss caused by low quality milk and poor yield from sub-clinical cows, treatment of affected animals, withdrawal from the milk line, and occasional culling of ill animals is not estimated. Accordingly, there is a need in the art for rapid, reliable and accurate tests for detecting mastitis.